Hygromycin-resistant transgenic plants

ABSTRACT

The present invention is directed to hygromycin-resistant transgenic plants and methods for producing such plants. The transgenic plants of the invention comprise gene constructs which encode and express hygromycin phosphotransferase or functional portion thereof.

This application is a continuation of application Ser. No. 07/586,317filed Sep. 19, 1990, now U.S. Pat. No. 6,048,730, which is acontinuation of application Ser. No. 07/169,560 filed Mar. 17, 1988,abandoned, which is a continuation of application Ser. No. 06/685,824filed Dec. 24, 1984, abandoned, the disclosures of all of which areincorporated herein in their entirety.

BACKGROUND OF THE INVENTION

Crown gall formation on dicotyledonous plants by Agrobacteriumtumefaciens is the result of the transfer and covalent integration of asmall segment called transfer DNA (T-DNA) of a tumor-inducing (Ti)plasmid into the chromosomal DNA of plant cells. The transferred T-DNAis expressed in plant cells and codes for several polyadenylatedtranscripts. Some of the transcripts are known to be responsible foropine synthesis and for tumor growth; these latter transcripts areencoded by oncogenes. None of these transcripts have been found to beessential for T-DNA transfer. The transfer mechanism is thought toinvolve repeated nucleotide sequences present near the T-DNA borders. Aslong as these borders are present, a foreign gene can be inserted intothe T-DNA of a Ti plasmid and thus engineered into the genome of a tumorcell or regenerated plant.

In addition to the T-DNA sequences, it is generally believed thatanother set of Ti plasmid genes, located outside the T-DNA, termed theVIR (virulence) region, play a role in the mobilization of the T-DNAfrom the bacterium to the plant cell. Heretofore, the presence of thecrown gall (tumor) on plants infected by an Agrobacterium carrying boththe T-DNA and VIR regions has been the primary means of identifyingtransformed plant cells. This means of identification has limitedcommercial utility however, because whole plants can not be regeneratedfrom crown galls containing functional oncogenes. Thus, it would beadvantageous to develop a way of introducing and expressing foreigngenes in plant cells without relying on tumor genes for the selectionand identification of transformed plant cells.

Presently, a variety of methods to introduce DNA into plant cells areavailable with varying degrees of success. These methods include the useof liposomes to encapsulate one or more DNA molecules, the contacting ofplant cells with DNA (which is complexed with either polycationicsubstances or calcium phosphate) and protoplast fusion techniques.Currently, the preferred technique involves the utilization of Tiplasmids from Agrobacterium cells to transfer a desired gene into aplant cell. Recently, researchers at Monsanto Company have demonstratedthe availability of a co-integrant Ti plasmid for use in a method fortransforming plant cells. (See, Fraley and Rogers, PCT ApplicationWO84/0219). In addition, a Ti binary vector system, developed by Hoekemaet al., 1983, Nature 303:179, is known in the art.

The aforementioned T-DNA region from the Ti plasmid is available for theinsertion of a desired gene which is under the control of a functionalplant expression mechanism. Such chimeric genes are known in the art toexpress both plant- and bacterial-derived polypeptides. Prior to thepresent invention, a chimeric protein, that is, a heterologous genewhich is fused to a portion or whole of a structural plant gene, hadnever been expressed in a plant cell. The present vector constructionsprovide for the production of such a chimeric protein and thuscontributes to the continuing development of plant transformationsystems.

As demonstrated for bacterial and mammalian cells, one of the primarysteps in the development of efficient transformation systems is theconstruction of dominant selectable markers. Such markers allow cellsthat have acquired new genes via transformation to be selected andidentified easily. The present invention provides novel expressionvectors which demonstrate that the aminocyclitol antibiotic hygromycin Bcan be the basis of such a selection scheme for transformed plant cells.

The present invention further provides a method of selecting transformedplant cells from a background of nontransformed cells. The method allowsone to add non-selectable DNA to the present vectors, transform plantcells with the modified vectors and select hygromycin resistanttransformants containing this otherwise non-selectable DNA. Sincetransformation is a very low frequency event, such a functional test isa practical necessity for determining which cell(s), of among millionsof cells, has acquired the transforming DNA.

For purposes of the present invention, as disclosed and claimed herein,the following terms are defined below.

Recombinant DNA Cloning Vector—any autonomously replicating agent,including but not limited to plasmids, comprising a DNA molecule towhich one or more additional DNA segments can or have been added.

Recombinant DNA Expression Vector—any recombinant DNA cloning vectorinto which one or more transcriptional and translational activatingsequence(s) have been incorporated.

Promoter—the site on the DNA molecule to which RNA polymerase attachesand at which transcription is initiated.

Amino terminal region-encoding sequence—the region of DNA at whichtranslation of mRNA into a polypeptide is initiated and a portion of the5′ end of the resulting polypeptide is produced.

Chimeric protein—a recoverable heterologous polypeptide which issynthesized from a gene containing a promoter and a portion of ahomologous coding region.

Border Sequence—DNA sequence which contains the ends of the T-DNA.

Broad-host-range replicon—a DNA molecule capable of being transferredand maintained in many different bacterial cells.

Conjugation—the process whereby DNA is transferred from bacteria of onetype to another type during cell-to-cell contact.

Crown Gall—a plant tumor caused by Agrobacterium tumefaciens. Tiplasmid—a large Agrobacterium plasmid which confers the ability toinduce tumors and promotes bacterial conjugation.

Micro-Ti Plasmid—a plasmid capable of replication in Agrobacteria andcontaining DNA flanked by T-DNA borders.

Non-oncogenic Strain—a strain of Agrobacterium tumefaciens which isunable to induce crown gall but retains the vir functions.

Transformations—the introduction of DNA into a recipient host cell thatchanges the genotype and results in a phenotypic change in the recipientcell.

SUMMARY OF THE INVENTION

In accordance with this invention, functional and selectable micro-Tiplasmids are disclosed. The hygromycin phosphotransferase (aphIV) genefrom Escherichia coli was inserted between the 5′ promoter andassociated amino terminal region-encoding sequence of an octopinesynthetase gene and the 3′ terminator sequence of a nopaline synthetasegene. These constructs were assembled between T-DNA border fragments ina broad-host-range vector to form micro-Ti plasmids of the presentinvention.

The invention further provides a method for selecting ahygromycin-resistant recombinant-DNA containing plant cell. The methodcomprises:

a) conjugating a recombinant DNA expression vector of the presentinvention into an Agrobacterium tumefaciens strain;

b) inoculating a hygromycin-sensitive plant cell with said Agrobacteriumtumefaciens containing the expression vector; and

c) selecting cells transformed by said expression vector underhygromycin resistance selective conditions.

BRIEF DESCRIPTION OF THE FIGS.

FIG. 1 shows the restriction site and function map of plasmid pCEL30.

FIG. 2 shows the restriction site and function map of plasmid pCEL40.

FIG. 3 shows the restriction site and function map of plasmid pCEL44.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a recombinant DNA expression vectorwhich is functional and selectable in plants comprising:

1) a transcription unit, flanked by transfer-DNA border sequences, whichcomprises a promoter and associated amino terminal region-encodingsequence and a terminator signal sequence,

wherein said sequences are derived from one or more genes which arenaturally expressed in a plant cell;

2) an antibiotic resistance gene encoding sequence located between saidpromoter and associated amino terminal region-encoding sequence and saidterminator sequence; and

3) a DNA fragment containing a replicon that is functional inAgrobacterium.

In accordance with the invention, an about 1.3 kb BamHI-BglII fragmentcoding for hygromycin phosphotransferase was introduced, in bothorientations, into a unique BglII restriction site of the intermediarycloning vector, plasmid pCEL30. Plasmid pCEL30 carries the promotertranscription signal and sequences coding for the first 11 amino acidsof the ocs gene. The introduction of the hygromycin phosphotransferase(aphIV) encoding gene was engineered to maintain the reading frame ofthe octopine synthetase (ocs) coding region. Thus, the aphIV gene wassubcloned into the BglII site of pCEL30, resulting—because of thespecific sequences involved—in the generation of a BglII-BamHI fusionnear the initiation codon of ocs. The resulting vector, plasmid pCEL40,encodes an ocs-aphIV fusion protein. When the aphIV gene was inserted inthe opposite orientation, hygromycin phosphotransferase was notproduced. A restriction site and function map of plasmids pCEL30 andpCEL40 is presented in FIGS. 1 and 2 of the accompanying drawings.

Plasmid pCEL30 can be conventionally isolated from Escherichia coli K12RR1ΔM15/pCEL30, a strain deposited and made part of The NorthernRegional Research Laboratory, Peoria, Ill. 61604. Plasmid pOW20, asource of the hygromycin phosphotransferase gene, is also deposited withthe NRRL and can be readily isolated from E. coli JA221/pOW20. Bothstrains are avilable to the public, as a preferred source and stockreservoir of the plasmids, under the accession numbers NRRL B-15915 andNRRL B-15838, respectively.

Since plasmid pCEL40 is not capable of replication in Agrobacterium, themicro T-DNA of plasmid pCEL40 was first transferred, as an EcoRIfragment, into a broad-host-range vector pKT210. This host vector isavailable from the Plasmid Reference Center, Stanford University, PaloAlto, Calif. 94305. The use of a specific host-range vector is notcritical in the construction of the present plasmids. Other such usefulvectors include, for example, pRK290 (Ditta et al., 1980, Proc. Natl.Acad. Sci. 77:7347-7351 and R772 (Hoekema et al., 1983, Nature303:179-180). If properly assembled and inserted into a plant genome, afusion protein will be expressed in the plant cell to create a desiredpolypeptide, such as a bacterial enzyme which confers antibioticresistance upon the plant. Thus, the resulting micro-Ti plasmid pCEL44is extremely useful because it contains a selectable and functionalplant gene for hygromycin resistance. In conjunction with the presenceof a selectable and functional plant gene, the unique construction ofplasmid pCEL44 confers additional beneficial attributes for use in planttransformation systems. For example, the T-DNA border sequences assistin the transfer and covalent integration of the transcription unit ofthe present micro-Ti plasmid into the chromosomal DNA of plant cells;the Agrobacteria-functional replicon provides an efficient means ofplasmid replication and eliminates a co-integration step used by othersskilled in the art; and the unique SalI cloning site provides aconvenient cleavage site for insertion of a gene of interest.

The above-illustrated vector confers hygromycin resistance to plantcells because of the presence of a functional phosphotransferase aphIVgene. Although the particular phosphotransferase gene inserted in theabove vector is an about 1.3 kb BamHI-BglII restriction fragment, otherknown phosphotransferase aphIV genes can be substituted. Such genesinclude, but are not limited to, those disclosed by Rao et al., 1983,Antimicrobial Agents and Chemotherapy 24:689-695. In addition, a varietyof different plasmid pOW20 restriction fragments that contain thehygromycin resistance-conferring gene can also be substituted providedthat any of these restriction fragments are positioned such that thepromoter region causes the transcription of the structural sequence. Thephosphotransferase aphIV gene is encoded on the about 1.3 kb BamHI-BglIIfragment of plasmid pOW20, therefore any restriction fragment containingthe afore-mentioned about 1.3 kb BamHI-BglII fragment also confers thedesired resistance to sensitive plant host cells. Skilled artisans willrecognize that all the above genes and fragments are functionallyequivalent and thus can be used and interchanged for purposes of thepresent invention.

Restriction fragments used to construct vectors illustrative of thepresent invention can be conventionally modified to facilitate ligation.For example, molecular linkers can be provided to a particularphosphotransferase aphIV-containing restriction fragment, to DNAcomprising the replication functions of the vector, or to DNA comprisingthe promoter or terminator sequences. Thus, specific sites forsubsequent ligation can be constructed conveniently. In addition, any ofthese DNA fragments can be modified by adding, eliminating orsubstituting certain nucleotides to alter characteristics and to provideor eliminate a variety of restriction sites for ligation of DNA. Thoseskilled in the art understand nucleotide chemistry and the genetic codeand thus which nucleotides are interchangeable and which DNAmodifications are desirable for a specific purpose.

The present invention also discloses a method for selecting ahygromycin-resistant recombinant DNA-containing plant cell, said methodcomprising:

1) conjugating the recombinant DNA expression vector of the presentinvention into an Agrobacterium tumefaciens strain;

2) inoculating a hygromycin-sensitive plant cell with said Agrobacteriumtumefaciens containing the expression vector; and

3) selecting cells transformed by said expression vector underhygromycin resistance selective conditions.

In accordance with the method of the invention, micro-Ti plasmids, suchas pCEL44, were conjugated into different strains of Agrobacteriumtumefaciens. Triparental matings were conducted with E. coli K12RR1ΔM15/pCEL44, an E. coli containing the helper plasmid pRK2013 (Dittaet al., supra) and A. tumefaciens LBA4013, a strain that contains awild-type Ti plasmid, pTiAch5. For purposes of this invention, the useof a specific helper plasmid or of a specific strain of A. tumefaciensis not critical. In this regard, any of the helper plasmids described inBagdasarian et al., 1981, Gene 16: 237-247 can be substituted forplasmid pRK2013, as the sole function of this kanamycin-resistant helperplasmid pRK2013 is to trans-complement the micro-Ti plasmid formobilization. In like manner, any oncogenic strain of A. tumefacienscarrying a wild-type Ti plasmid with a functional vir region, which iscapable of transferring its own Ti plasmid to a plant cell andtransforming the plant cell, can be used in the present invention.Various strains of Agrobacterium tumefaciens that are suitable for usein the present invention are publicly available; see, e.g., ATCCCatalogue of Strain I, p.66 (15th edition, 1982).

A preferred embodiment of the invention utilizes a non-oncogenic, oravirulent, strain of A. tumefaciens. Such a strain can be constructedthrough the receipt of a Ti-plasmid from which the T-DNA region has beendeleted. A particular strain of Agrobacterium tumefaciens available foruse in the present invention is A. tumefaciens LBA-4404. The strain wasdeveloped by Dr. P. J. J. Hooykaas and is deposited in the CentraleCollectie van Schimmel-cultures (CBS) at Baarn as number CBS191.83. Thisstrain is also deposited and made part of the Northern Regional ResearchLaboratory and is available to the public under the accession numberNRRL B-15920. Plant cells exposed to this strain of bacteria do not formcrown galls. Instead, hygromycin resistant callus is produced which iseasily regenerated into mature plants.

The infection of plant tissue by Agrobacterium is a simple techniquewell known to those skilled in the art. Typically, a plant is wounded byany of a number of ways, which include cutting with a razor, puncturingwith a needle, or rubbing with abrasive. The wound is then inoculatedwith a solution containing tumor-inducing bacteria. In the presentinvention, Agrobacteria containing a binary vector system consisting ofthe wild-type Ti plasmid and the micro-Ti plasmid pCEL44 were used toincite galls on asceptic decapitated seedlings of Nicotiana tabacum cvWisconsin 38. In addition, a non-oncogenic strain of A tumefacienscontaining the micro-Ti plasmid pCEL44 was used to induce callusproduction from leaf sections of Nicotiana plumbaginifolia and Nicotianatabacum cv Wisconsin 38. Both of these varieties are readily availablefrom the United States Department of Agriculture's Tobacco ResearchLaboratory, Box 16G, Oxford, N.C. 27565. The use of a cell from aspecific type of plant is not critical to the present invention since acell from any plant, into which a Ti plasmid can be transformed byAgrobacteria, can be utilized. For example, the cell can come from anydicotyledonous plant, such as, tomato, potato, tobacco, sunflower andsoybean or from a monocotyledonous plant, such as members of thefamilies, Liliaceae and Amaryllidaceae.

The resultant tumorous growths were excised and DNA was isolated fromrepresentative tobacco clones, digested with BamHI and BamHI plusHindIII restriction enzymes and Southern-blotted. The about 1.3 kbBamHI-BglII fragment conferring hygromycin resistance wasnick-translated for use as a hybridization probe for analysis of theT-DNA structure.

A plant cell, transformed with a Ti plasmid in accordance with thisinvention, is used to regenerate a plant that expresses hygromycinresistance, as well as other functions, for which the Ti plasmid codes.The invention is useful for genetically modifying plant tissues andwhole plants by introducing useful plant genes from other plant speciesor strains, such useful plant genes include, but are not limited to,genes coding for storage proteins, lectins, resistance factors againstdisease, insects and herbicides, factors providing tolerance toenvironmental stress and the like. The method, plasmids andtransformants of the present invention provide plant breeders with anovel way of introducing desirable genes into plants, as well as toprovide plant molecular biologists with molecular probes for studyingplant development.

The examples, which follow, further illustrate this invention.

EXAMPLE 1 Culture of E. Coli RR1ΔM15/pCEL30 and Isolation of PlasmidpCEL30.

A. Culture of E. coli RR1ΔM15/pCEL30

E. coli RR1ΔM15/pCEL30 (NRRL B-15915) was grown in 750 ml of L medium(10 g/l caesin hydrolysate, 5 g/l yeast extract, 5 g/l NaCl, 1 g/lglucose, pH 7.4) containing ampicillin at 50 μg/ml according toconventional microbiological procedures. The culture was harvested after24 hours incubation at 37° C. with vigorous shaking.

B. Isolation of Plasmid pCEL30

The culture was centrifuged and the cell pellet resuspended in 50 mlfreshly-prepared lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 9 mg/mlglucose, 2 mg/ml lysozyme). After 45 minutes incubation on thesuspension was mixed with 100 ml of a solution that was 0.2N NaOH and 1%SDS and then kept on ice for a further 5 minutes. Another 90 ml of 3Msodium acetate was added and the mixture maintained on ice for 60minutes.

Cell debris was removed by centrifugation and the supernatant was mixedwith 500 ml ethanol. After 2 hours at −20° C., nucleic acid was pelletedby centrifugation and resuspended in 90 ml of 10 mM Tris-HCl pH 8, 10 mMEDTA.

The nucleic acid solution was mixed with 90 gm cesium chloride and 0.9ml of a solution containing 10 mg/ml of ethidium bromide, thencentrifuged at 40,000 rpm for 24 hours to purify the plasmid DNA. Theplasmid DNA band was recovered and recentrifuged at 40,000 rpm for 16hours. The plasmid DNA band was again recovered and freed of cesiumchloride and ethidium bromide by conventional procedures andprecipitated with 2 volumes of ethanol containing 90 g/l ammoniumacetate. The pelleted DNA was dissolved in TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA) at a concentration of 0.2 mg/ml.

EXAMPLE 2 Culture of Escherichia coli JA221/pOW20 and Isolation ofPlasmid pOW20

A. Culture of E. coli JA221/pOW20

This bacterium is grown as described for E. coli RR1ΔM15 in Example 1above.

B. Isolation of Plasmid pOW20

This plasmid is prepared as described for plasmid pCEL30 in Example 1above.

EXAMPLE 3 Construction of Escherichia coli RR1ΔM15/pCEL40

A. BglII Digestion of Plasmid pCEL30 and Treatment With Calf IntestinalPhosphatase

Five μg of plasmid pCEL30 DNA were digested with 50 units of BglIIrestriction enzyme in a 150 μl reaction of the composition recommendedby the enzyme manufacturer*. Digestion was allowed to proceed for 90minutes at 37° C. *Restriction and other enzymes can be readily obtainedfrom the following sources:

Bethesda Research Laboratories, Inc. Box 6010 Rockville, Md. 20850

Boehringer Mannheim Biochemicals 7941 Castleway Drive P.O. Box 50816Indianapolis, Ind. 46250

New England Bio Labs., Inc. 32 Tozer Road Beverly, Mass. 01915

B. BamHI-BglII Digestion and Isolation of the about 1.3 kb Fragment ofPlasmid pOW20

The reaction was first mixed with 8.75 μl of 0.5M Tris-HCl pH 8, 1 mMEDTA and then with 1.25 units of calf intestinal phosphatase (BoehringerMannheim) and incubated at 37° C. for 15 minutes. The mixture wasextracted with buffered phenol, then with ether and precipitated with 2volumes of ethanol containing ammonium acetate. After 30 minutes at −70°C., the DNA was pelleted and redissolved in TE buffer at a concentrationof 10 μg/ml.

About 20 μg of plasmid DNA is digested with the restriction enzymesBamHI and BglII according to the enzyme manufacturer's recommendedprocedures.

The DNA fragments resulting from this digestion are fractionated byconventional methods of agarose gel electrophoresis and isolated byentrapment on a piece of NA-45 DEAE paper (Schleicher & Schuell Inc.,Keene, N.H. 03431) inserted into the gel during electrophoresis. DNA iseluted from the paper by spinning the paper for 5 seconds with asufficient amount of a high salt buffer (1.0M NaCl; 0.1 mM EDTA; and 20mM Tris, pH 8.0) to cover the paper in a micro-centrifuge. The paper isincubated at 55° C. -60° C. for 10-45 minutes with occasional swirling.The buffer is removed and the paper washed with about 50 μl of buffer.The DNA is extracted first with phenol and then with ether precipitatedwith two volumes of ethanol containing ammonium acetate, and resuspendedin TE buffer at a concentration of about 25 μg/ml.

C. Ligation

Ten ng of the phosphatased, BglII-cut plasmid pCEL30 were mixed with 50ng of the purified about 1.3 kb BamHI-BglII fragment in a 15 μl ligasebuffer (50 mM Tris-HCl, pH 7.6; 10 mM MgCl₂; 10 mM DTT; and 1 mM ATP)containing 0.8 units of T4 DNA ligase (BRL). The mixture was incubatedovernight at 15° C.

D. Transformation of E. coli RR1ΔM15 and Selection

The ligation mixture was mixed with 15 μl sterile 60 mM CaCl solution.Next, 70 μl of a suspension of competent E. coli RR1ΔM15 cells, whichhad been stored 20× concentrated in 30 mM CaCl, 15% glycerol at −70° C.,were added. After 60 minutes on ice, the transformation mixture washeat-treated at 42° C. for 2 minutes and then incubated with 0.5 ml Lmedium for 90 minutes at 37° C.

Samples of mixture were spread on L medium containing ampicillin at 50mg/l and solidified with agar at 15 g/l. These samples were thenincubated overnight at 37° C. to permit growth of colonies fromtransformed cells.

E. Identification of Clones of E. coli RR1ΔM15Containing Plasmid pCEL40

Colonies resulting from the transformation were inoculated into 5 ml Lmedium containing ampicillin at 50 mg/ml and grown overnight at 37° C.Plasmid DNA was prepared from 1 ml samples of these cultures by theprocedure of Holmes & Quigley (Analytical Biochemistry 114:193; 1981)and redissolved in 50 μl of TE buffer.

The plasmids were digested in 10 μl reactions containing 7.5 μl of theDNA solution and about 5 units each of HindIII, PstI, BglII and BglIIplus HindIII restriction enzymes and other reagents as suggested by theenzyme manufacturers. After 60 minutes at the appropriate temperaturethe digests were analysed by agarose gel electrophoresis usingconventional procedures. The sizes of fragments produced from pCEL40 byrestriction enzymes were consistent with the plasmid structure in FIG.2.

EXAMPLE 4 Construction of Micro-Ti Plasmid pCEL44

A. EcoRI Digestion of Plasmid pKT210 and Phosphatase Treatment

Five μg of plasmid pKT210 were digested with 50 units of EcoRIrestriction enzyme in a 150 μl reaction of a composition recommended bythe enzyme manufacturer. After 90 minutes of 37° C., the reaction wastreated with calf intestinal phosphatase as described in Example 3 anddissolved in TE buffer at a concentration of 10 μg/ml.

B. EcoRI Digestion of Plasmid pCEL40

Fifteen μl of a preparation of plasmid pCEL40 DNA, constructed inExample 3E, were digested with 10 units of EcoRI restriction enzyme in a20 μl reaction at 37° C. for 90 minutes and then extracted with phenol,followed by an ether extraction. The digested DNA was precipitated with2 volumes of ethanol containing ammonium acetate at −20° C. andredissolved in 20 μl TE buffer.

C. Ligation, Transformation and Selection of E. coli RR1ΔM15/pCEL44

Ten ng of phosphatased, EcoRI-cut pKT210 were ligated with 5 μl ofEcoRI-cut pCEL40 as described in Example 3C, and transformed into E.coli RR1ΔM15 as described in Example 3D.

Transformed cells were selected by their ability to grow on solidified Lmedium containing chloramphenicol at 10 mg/l Colonies containing pCEL44were identified by PstI, HindIII and BamHI restriction enzyme analysisof their constituent plasmids as described in Example 3E. A restrictionsite and function map is presented in FIG. 3 of the accompanyingdrawings.

EXAMPLE 5 Conjugation of pCEL44 Into Agrobacterium tumefaciens LBA4013

A. Growth of Parental Strains

Escherichia coli K12 RR1ΔM15/pCEL44 and E. coli pRK2013 were grownovernight at 37° C. on solidified L medium. Agrobacterium tumefaciensLBA4013 was grown for 2 days at 28° C. on solidified L medium.

B. Triparental Conjugation and Selection

One loop of each of the three strains described above were mixed in 1 mlof 30 mM magnesium sulfate solution. Next, a drop of the mixture wasplaced on solidified TY medium (5 g/l caesin hydrolysate, 5 g/l yeastextract, 15 g/l agar) and incubated at 28° C. overnight.

The bacterial growth was resuspended in 3 ml of 10 mM magnesium sulfatesolution and 0.1 ml samples of serial dilutions were spread onsolidified TY medium containing 100 mg/l nalidixic acid and 2 mg/lchloram-phenicol and incubated at 28° C.

Transconjugants gave rise to individual colonies after 2 to 4 daysgrowth. These were inoculated singly into 25 ml liquid TY mediumcontaining 100 mg/l nalidixic acid and 2 mg/l chloramphenicol andincubated at 28° C. with shaking for another 2 days. The plasmid contentof the transconjugants was then checked by the method of Casse et al.(Journal of General Microbiology 113:229-242; 1979).

EXAMPLE 6 Introduction and Expression of Hygromycin Resistance inTobacco Crown Gall Cells

Aseptic tobacco plants of cultivar Wisconsin 38 were raised as follows.Seed was sterilized by a 10 minute treatment with 10% Clorox bleach,followed by a rinse with 95% ethanol and then extensive rinsing withsterile water. Seed was planted on the surface of medium R5, whichconsists of MS major and minor salts (Physiol. Plant. 15:473-497, 1962),with 30 g/l sucrose, 100 mg/l myo-inositol (Sigma), 2.5 mg/l thiamineHCl (Eli Lilly and Company), 2.5 mg/l nicotinic acid (Matheson ColemanBell), 1.25 mg/l pyridoxine HCl (Lilly), and 1.25 mg/l Ca pantothenate(Sigma), pH 6, solidified with 9 g/l Difco Bacto agar. Germinated seedswere picked onto the same R5 medium and the plants were maintained bymaking vegtative cuttings approximately every 2 months into Magenta GA7boxes. They were grown in a lighted incubator with a day length of 16hours, light intensity of about 150 lux, and a temperature of 26° C.

To inoculate with Agrobacterium, the sterile plants were decapitated anddefoliated with a sterile scalpel, leaving a large wound at the apex andthe cut petioles extended. Within a few minutes, a culture ofAgrobacteria tumefaciens LBA 4013/pCEL44 was applied to cut surfacesuntil a cream-brownish accumulation was visible to the naked eye. Theinoculated, wounded plants were labeled, sealed in boxes, and placedback into the lighted incubator. Care was taken to insure that ambienttemperature was never over 30° C. during gall induction.

Fifteen days after inoculation, visible galls had grown out of mostwound inoculation sites on the sterile Wisconsin 38 plants. These gallswere cut off individually, numbered, and placed onto R5 medium which hadbeen supplemented with 200 μg/ml each carbenicillin (Sigma) andvancomycin (Vancocin HCl Lilly). This treatment, intended to kill thetumor-inciting bacteria and give aseptic cultures, involved culturingthe crown galls in the dark at 25° C. to 27° C. for three weeks on theabove antibiotics, followed by another three-week passage on thoseantibiotics.

To test the response to hygromycin of these crown gall callus tissuecultures, 100 mg callus samples were placed onto Falcon 1007 petridishes filled with about 15 ml of callus growth medium containing MSsalts, 30 g/l sucrose, 1 mg/l thiamine HCl, 100 mg/l myo-inositol, 9 g/lDifco Bacto-agar, 1 mg/l IAA (indole-3-acetic acid, Sigma), 0.1 mg/lkinetin (6-furfurylaminopurine, Sigma), pH 6. After these samples weresterilized, the aforementioned concentrations of vancomycin andcarbenicillin, and 50 μg/ml of hygromycin B (Lilly) were added. Thistest was read after three weeks of incubation in the dark at 27° C. andpositive growth phenotypes recovered.

EXAMPLE 7 Presence of aphIV Sequences in Hygromycin-Resistant GallTissues

A. Southern Blot of Plant DNA

Nuclear DNA was prepared from about 10 gm of gall tissue by the methodof Rivin et al. described in “Maize for Biological Research” pp. 161-164(ed W. F. Sheridan, publ. Plant Molecular Biology Assn; Charlottesville,Va., 1982).

Ten μg of each DNA was digested with 100 units each of BamHI and BamHIplus HindIII restriction enzymes in a 200 μl reaction of the compositionrecommended by the enzyme manufacturer. After 4 h incubation at 37° C.,the digested DNA was precipitated with 400 μl of ethanol containingammonium acetate for 30 minutes at −70° C. The DNA was allowed todissolve overnight at 4° C. in 25 μl of TE buffer.

The digested DNA fragments were fractionated by conventional agarose gelelectrophoresis and transferred to GeneScreen, a hybridization transfermembrane (New England Nuclear, Boston, Mass. using the manufacturer'sprotocol.

B. Probe from the 1.3 kb Fragment of Plasmid pOW20

A hybridization probe is made from the fragment of plasmid pOW20purified as in Example 3B. The fragment is nick-translated byconventional procedures (see Maniatis et al., 1982 Molecular Cloning,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) using DNase Iand DNA polymerase such that the probe contains about 20 μCi of ³²P-dCTPper 0.2 μg of DNA.

C. Hybridization to Detect aphIV Sequence Homology

GeneScreen containing plant DNA fragments was prehybridized overnight at42° C. in a solution containing 0.75 M NaCl, 75 mM sodium citrate, 25 mMsodium phosphate pH 6.7, 2 g/l Ficoll 400, 2 g/l polyvinylpyrrolidone360, 2 g/l bovine serum albumen, 1% sodium dodecyl sulfate, 10% dextransulfate, 50% formamide and 250 mg/l denatured, sonicated calf thymusDNA.

Hybridization was in fresh solution of the same composition butsupplemented with heat-denatured probe DNA (0.2 μg per 25 ml ofsolution). After overnight incubation at 42° C., the GeneScreen membranewas washed twice with GS buffer (0.3 M NaCl, 60 mM Tris-HCl, 2 mM EDTA,pH 8). Each wash was performed for 1 hour at room temperature. Themembrane was then washed stringently with 0.1×GS buffer supplementedwith 1% SDS for 1 hour at 70° C. This step was repeated and then theGeneScreen was washed twice more at room temperature with 0.1×GS buffer.The membrane was blotted dry and exposed to X-ray film overnight at −70°C. with intensifying screens.

EXAMPLE 8 Regeneration of Plants

A. Regeneration from Uncloned Gall Tissue

Uncloned gall tissue which had demonstrated strong growth in thepresence of a restrictive level of hygromycin B was subjected to atreatment intended to induce plant regeneration under selectivepressure. This treatment comprised placing pieces of callus tissue on MSmedium with 0.3 mg/l IAA and 10 mg/l 2-ip (6-gamma, gamma-dimethylallylaminopurine, Gibco*) 100 mg/l myoinositol, 1 mg/l thiamine HCl, pH 6, 30g/l sucrose, solidified with 0.9% Difco Bacto-agar plus 50 μg/ml ofhygromycin. These preparations were placed in a lighted incubator forone month and then dark green and budding areas of tissue were selectedand transferred to the same high-cytokinin, hygromycin-supplementedmedium again for another month of incubation in the light. Followingthese two passages on regeneration medium plus antibiotic, any green,budding clumps of plantlets were cut to excise single plants which werefurther cultured on solid medium R5 for sterile plant growth in thelight. When well-developed plants were obtained from three differenttissue lines, the expression of the hygromycin resistance gene in theseplants was tested as follows. Leaf pieces about a cm square were cut outand placed onto callus growth medium with or without hygromycin.

* Plant media and supplements can be readily obtained from Gibco, GrandIsland, N.Y.

Control tissues were leaf pieces from the aseptic Wisconsin 38 plantswhich were starting material for these crown gall induction experiments.These preparations were incubated in the dark at 27° C. After threeweeks, the Wisconsin 38 and the regenerated gall tissue plants bothshowed leaf cell enlargement and callus growth at the edges of the leafpiece explants in antibiotic-free medium. In the antibiotic-containingmedium, however, the control Wisconsin 38 leaf material was unable toinitiate cell division and was brown and dying, while the leaves fromplants regenerated under selection pressure from hygromycin-resistantgall cultures showed a cell expansion, growth and callus productioncomparable to the no-hygromycin dishes. These results indicate that theregenerated plants contain and are capable of expressing the hygromycinresistance gene from pCEL44.

B. Single Cell-Derived Clones from Hygromycin Resistant Galls

Hygromycin-resistant crown gall lines were maintained in the dark at 27°C. without antibiotics, with monthly subcultures on medium 1/0.1 (MSsalts with 1 mg/l IAA and 0.1 mg/l kinetin, 0.9 g/l Difco Bacto agar,100 mg/l myo-inositol, 1 mg/l thiamine HCl, 30 g/l sucrose, pH 6,autoclaved to sterilize and dispensed 50 ml/dish into Falcon 1005 petridishes). Samples from these culture lines were dispersed into the samemedium, which had been prepared without agar and dispensed 50 ml/flaskin 125-ml erlenmeyer flasks and plugged with Jaece foam stoppers.Suspension cultures initiated in this way were shaken at 135 rpm in agyrotory shaker in the dark at 27° C. After a week, the hard clumps ofcallus were removed and the suspension cultures of crown gall cells werepassaged by dividing 1:1 into fresh liquid medium on a weekly basis.These short-term suspensions can also be cultured for a month beforereverting to callus clumps in the shake cultures.

To generate single-cell-derived clones, protoplasts were prepared fromthese cultures and then cultivated at extremely low population density,as follows. The short-term suspension cultures were centrifuged lightlyto concentrate the cells; the growth medium in the supernatant wasremoved, and 5 ml packed cell volume of the cells was mixed with 25 mlof a filter-sterilized enzyme mixture consisting of 6% w/v cellulysin(Onozuka R10, Kinki Yakult Mfg., Japan), 1% w/v macerase (MacerozymeR10, also from Kinki Yakult), 9% w/v mannitol (Sigma), 3 mM MES(2-(N-morpholine)-ethane sulfonic acid, Sigma), pH 5.8. The cell plusenzyme mixture was placed in a Falcon 1005 petri dish and incubated{fraction (41/2)} hours at room temperature with slow rotary shaking at50 rpm. When microscopic observation showed protoplast release, thispreparation was filtered through wire mesh screens of aperture size 259and 231 microns (W.S. Tyler, Inc. of Mentor, Ohio), and mixed 1:1 withprotoplast culture medium which had been made up to 25% w/v Ficoll DL(Sigma). This mixture was placed in the bottom of a round-bottom glasscentrifuge tube and overlayered with a few ml of the same protoplastculture medium made up to 8% w/v Ficoll, and finally, with a thin 1-2 mllayer of the protoplast culture medium made up to 2% w/v Ficoll. AllFicoll-containing solutions had been filter-sterilized. Thesediscontinuous density gradient preparations were capped and centrifugedin a swinging bucket, ambient-temperature centrifuge at 50×x G for 30minutes. The protoplasts were layered at the top and collected with awide-bore pasteur pipet at high population density, 2×10⁵ viable cellsper ml in this experiment. The protoplast culture medium used was KP(medium K3 of Kao & Michayluk, Planta 120:215-227, 1974, as modified byCaboche in Muller et al., Physiol. Plant. 57:35-41, 1983), made up to anosmotic strength of 0.5M with mannitol.

Protoplasts thus collected were cultured at the high population density(as collected), in aliquots of 1 ml in Falcon 3001 dishes, in the darkat 26° C. After six days, they were examined for growth, cell viabilityas determined by exclusion of the vital stain Evans blue (Sigma), andcell wall formation and divisions. Their diameter was measured under themicroscope. The preparation was filtered through a 107 microns diameterwire mesh filter. The preparation was checked for the absence of cellaggregates. The cells then were serially diluted, with populationdensity counting, to a final population density of about 100 viablecells per ml in medium C of Caboche, supra which had been adjusted tothe same osmotic strength as the culture medium, using mannitol. About15 ml per dish were plated and placed in the lighted incubator.Single-cell-derived colonies were obtained in a month with somecultures, and after two months with other cultures. These colonies werepicked from the liquid medium with a wide-bore pasteur pipet and placedon a Whatman #3 filter paper soaked in liquid medium 1/0.1. Thesepaper-supported cultures were continued in the light for another monthwith additions of the liquid medium to the paper when needed, followedby picking onto solid medium 1/0.1 and cultured in the dark at 27° C.

In a test for the expression of hygromycin resistance in singlecell-derived callus cultures, paired samples of cloned lines of callustissue were plated on solid medium 1/0.1 with 50 μg/ml hygromycin B, andon the same medium without antibiotic. After 3 weeks of culture in thedark at 27° C., the two dishes were compared. In 52 out of 56 cases, thecalli exhibited hygromycin resistance by growing as well with the 50μg/ml hygromycin B as without.

C. Regeneration from Cloned Tissue

The hygromycin-resistant callus tissue cultures derived from theprotoplast method of single cell cloning in section 8B can be subjectedto a regenerating protocol. Callus pieces are placed on solid mediumcontaining MS salts, 100 mg/l myo-inositol, 1 mg/l thiamine HCl, 0.3mg/l IAA, 10 mg/l 2-ip, 0.9 g/l Difco Bacto-agar, 30 g/l sucrose, pH 6and cultured in the light for one month. Green and budding pieces oftissue are passaged again on the same medium. Plantlets formed underthis regime are passaged onto medium R5, described in section 6 above,for development into well-formed plants in the light. The plantsobtained are tested for the expression of the hygromycin resistance genefrom plasmid pCEL44 by incubating leaf piece explants in the dark oncallus growth medium containing hygromycin, as described in section 8Aabove. The plants demonstrate expression of the hygromycin resistancegene by showing leaf cell expansion and callus formation on the cutedges of the leaf piece explants, as in section 8A.

These plants are transplanted into soil and brought to sexual maturityin the greenhouse so that Mendelian inheritance of the hygromycinresistance gene is demonstrated. This proves that the binary vectorcontaining pCEL44 can transfer only the hygromycin resistance genewithout the tumor-inducing oncogenes, providing a new and useful way toengineer desired traits into plant cells which can be regenerated intosexually competent plants.

EXAMPLE 9 Conjugation of pCEL44 into Agrobacterium tumefaciens LBA4404

A triparental cross between E coli K12 RR1ΔM15, E. coli pRK2013 and A.tumefaciens LBA4404 (NRRL B-15920) was performed as described in Example5. Transconjugants were selected on solidified TY medium containing 20mg/l rifampicin and 2 mg/l chloramphenicol.

EXAMPLE 10 Introduction and Expression of Hygromycin Resistance inCallus

A. Nicotiana plumbaginifolia

Seeds of N. Plumbaginifolia were sterilized by a brief exposure toethanol followed by a 3 minute treatment with 0.5% Chlorox bleach. Afterrinsing with sterile water the seeds were incubated for 1 hour in asolution containing 0.5 mg/ml gibberillic acid. Next, the seeds wereplanted on MS medium solidified with 0.6% Phytagar. Plants were grown at27° C. with a daylength of 16 hours at a light intensity of about 700lux.

Leaf sections of about 1 sq cm were cut from 3 month-old plants and thecut edges were smeared with a culture of Agrobacterium tumefaciensLBA4404/pCEL44. The infected sections were incubated on solidified MSmedium containing 1 mg/l napthaleneacetic acid (NAA) and 0.1 mg/lbenzyladenine (BA) for 3 days in the dark at 27° C. The sections werethen transferred to the same medium supplemented with 200 mg/lvancomycin and 200 mg/l carbenicillin. After growth for one month,pieces of callus were transferred to MS medium with 1 mg/ml NAA, 0.1mg/ml BA and 50 mg/l hygromycin B and the incubation continued.

After 3 weeks, small pieces of callus were inoculated onto solidified MSmedium supplemented with 0.1 mg/l NAA and 1 mg/l BA and incubated at 27°C. in the light with a 16 hour day to regenerate shoots. The shoots aretransferred to solidified MS medium without supplements to promote rootgrowth.

B. Nicotiana tabacum

A portion of a tobacco callus suspension culture, NT575, was poured ontosolid media (described in Example 8C). After excess water was removed, afeeder layer system was constructed wherein two layers of sterilizedWhatman #1 filter paper were placed over the cells. The cells wereincubated in the dark at 26° C. overnight.

A culture of Agrobacterium tumefaciens LBA-404/pCEL44 was grown up on TYmedia with 2 mg/l chloramphenicol. Isolates were transferred into aliquid medium containing 5 g/l casein hydrolysate and 3 g/l yeastextract, and shaken overnight at 26° C. in a gyrotory shaker. Leafsections of about 1 sq cm were cut from 3 month-old plants and soaked inthe liquid overnight bacterial culture for 6-8 minutes, blotted dry andplaced onto the filter paper cover of the above prepared feeder layersystem.

The leaf pieces were incubated in the light at 26° C. for two days, andthen picked off and placed on regeneration medium plus 200 μg/ml eachcarbenicillin and vancomycin. After an additional 12 days in the lighton this medium, the leaf pieces were moved onto the same medium with 50μg/ml hygromycin. These plates were cultured in the light of 26° C. foran additional 3 months.

Plantlets were regenerated and grew out on the hygromycin-containingmedium from the treated leaf pieces. Pieces which had been treated inthe same way but with Agrobacteria containing the hygromycin resistancegene in an inverted orientation gave no plantlets.

Plants are picked from these leaf sections, grown in hormone-free R5medium and tested for hygromycin resistance by explanting leaf piecesonto callus growth medium containing 50 μg/ml hygromycin B insubstantial accordance with the teaching of Example 8A.

Similarly, leaf sections are treated as taught above, except that callusgrowth medium (Example 10A) is used in place of the regeneration medium.These leaf sections are grown in callus growth medium supplemented with200 μg/ml each vancomycin and carbenicillin. These preparations areincubated in the dark at 26° C. for 2 weeks and then moved to thesupplemental medium containing 50 μg/ml hygromycin B. Hygromycinresistance is proven by growing callus on antibiotic-containing mediaand by probing Southern blots of the plant DNA with the aphIV sequenceas taught in Example 7.

What is claimed is:
 1. A dicotyledenous transgenic plant comprising achimeric gene which comprises: (a) a plant-expressible promoter; and (b)a coding sequence of an aphIV gene encoding a functional portion of ahygromycin phosphotransferase enzyme, which coding sequence ispositioned 3′ of said promoter as to be expressible, wherein expressionof said sequence confers hygromycin resistance to said transgenic plantor cells thereof.
 2. The transgenic plant of claim 1, wherein, in the 3′direction from the promoter, and before the 5′ end of the codingsequence there is an amino terminal region-encoding sequence, whichamino terminal region-encoding sequence is under transcriptional controlof said promoter when the promoter is naturally occurring.
 3. Thetransgenic plant of claim 1, wherein the promoter is from an octopinesynthetase gene.
 4. The transgenic plant of claim 2, wherein the aminoterminal region-encoding sequence is from an octopine synthetase gene.5. The transgenic plant of of claim 1, wherein said chimeric geneadditionally comprises a terminator signal sequence.
 6. The transgenicplant of claim 1, wherein said terminator signal sequence is from anopaline synthetase gene.
 7. A plant part from the transgenic plant ofany one of claims 1-6.
 8. Seed from the transgenic plant of any one ofclaims 1-6.
 9. A method for producing a dicotyledenous transgenic plant,comprising regenerating said plant from a hygromycin resistantdicotyledenous transgenic plant cell, which transgenic plant cellcomprises (a) a plant-expressible promoter; and (b) a coding sequence ofan aphIV gene encoding a functional portion of a hygromycinphosphotransferase enzyme, which coding sequence is positioned 3′ ofsaid promoter as to be expressible, wherein expression of said sequenceconfers hygromycin resistance to said transgenic plant cell.
 10. Themethod according to claim 9, wherein, in the 3′ direction from thepromoter, and before the 5′ end of the coding sequence there is an aminoterminal region-encoding sequence, which amino terminal region-encodingsequence is under transcriptional control of said promoter when thepromoter is naturally occurring.
 11. The method according to claim 9,wherein the promoter is from an octopine synthetase gene.
 12. The methodaccording to claim 10, wherein the amino terminal region-encodingsequence is from an octopine synthetase gene.
 13. The method accordingto claim 9, wherein said chimeric gene additionally comprises aterminator signal sequence.
 14. The method according to claim 9, whereinsaid terminator signal sequence is from a nopaline synthetase gene.